Fractionation and characterization of the phosphoenolpyruvate: fructose 1-phosphotransferase system from Pseudomonas aeruginosa.
The initial reactions involved in the catabolism of fructose in Pseudomonas aeruginosa include the participation of a phosphoenolpyruvate:fructose 1-phosphotransferase system (F-PTS). Fractionation of crude extracts of fructose-grown cells revealed that both membrane-associated and soluble components were essential for F-PTS activity. Further resolution of the soluble fraction by both size exclusion and ion-exchange chromatography revealed the presence of only one component, functionally analogous to enzyme I. Enzyme I exhibited a relative molecular weight of 72,000, catalyzed the pyruvate-stimulated hydrolysis of phosphoenolpyruvate to pyruvate, and mediated the phosphorylation of fructose when combined with a source of enzyme II (washed membranes). No evidence for the requirement of a phosphate carrier protein, such as HPr, could be demonstrated. Thus, the F-PTS requires a minimum of two components, a soluble enzyme I and a membrane-associated enzyme II complex, and both were shown to be inducible. Reconstituted F-PTS activity was specific for phosphoenolpyruvate as a phosphate donor (Km, approximately -0.6 mM) and fructose as the sugar substrate (Km, approximately 18 microM). Components of the Pseudomonas F-PTS did not restore activity to extracts of deletion mutants of Salmonella typhimurium deficient in individual proteins of the PTS or to fractionated membrane and soluble components of the F-PTS of Escherichia coli. Similarly, membrane and soluble components of E. coli and S. typhimurium would not cross-complement the F-PTS components from P. aeruginosa.
D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000032:analysis / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012486:Salmonella typhimurium / Q000201:enzymology; D013045:Species Specificity; D013379:Substrate Specificity
D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D008274:Magnesium